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gene disruptions

Sandy Silverman sandy at icr.pt.cyanamid.COM
Tue Nov 15 17:03:45 EST 1994


In article <01HJ385VDPB48Y5H5P at mbcl.rutgers.edu> neigeborn at OCELOT.RUTGERS.EDU ("Neigeborn, Lenore") writes:

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   From: neigeborn at OCELOT.RUTGERS.EDU ("Neigeborn, Lenore")
   Newsgroups: bionet.molbio.yeast
   Date: 4 Nov 1994 14:20:27 -0800
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   Robin,
	I've made many one-step gene replacements using sites from polylinkers and
   I've never had a problem.  I've had as many as 30 bases of nonhomology on the
   ends.  The explanation I've heard is that there is considerable exonuclease
   activity nibbling at the ends of the linear fragment prior to the recombination
   event, so that by the time rec. occurs the nonhomology regions are eliminated.

	In a somewhat related mode, there is this new procedure for making gene
   replacements that involves PCR-ing a selectable marker using oligos containing
   homology flanking your locus on the 5' end and homology to the marker on the 3'
   end (thus when PCR is complete you have a fragment with homology to your locus
   on the ends and a selectable marker in the middle).  Then you transform in this
   fragment for a one-step replacement.  This allows you to make any deletion you
   choose (you're not a slave to your restriction sites). 

	 We've tried this several times with zero success.  We get transformants
   but they are all conversions at the marker locus (we assume, they are not at
   the desired locus).  Has anyone had decent success with this?  We've used
   oligos with 50 bases of homology to our locus on either end.  We have no
   trouble with the PCR reaction, just with the gene replacement.  We've tried
   URA3 and LEU2.

   Lenore Neigeborn
   Neigeborn at MBCL.rutgers.edu

RESPONSE FROM SANDY::

We've also had trouble with this.  I think the key is to use a strain with
a complete deletion of sequences of the marker (i.e. his3-delta-200, as in
the Lacroute paper).  The problem here is that the YPH499, YPH500 strains
that have that allele are known to act funny.  We are presently trying
other strains with this his allele.  If anyone has a really well behaved
one, I'd be interested. 

-------Sandy

--
Sanford Silverman                      >Opinions expressed here are my own<
American Cyanamid  
silvermans at pt.cyanamid.com     			"Yeast is Best"



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